G protein-coupled receptors (GPCRs) mediate hormonal control of numerous intracellular effectors. GPCR signaling systems are tightly regulated and rapidly attenuate their hormonal responsiveness following activation. While multiple processes contribute to this loss of responsiveness, one primary mechanism involves stimulus-dependent phosphorylation of the receptor. This is predominantly mediated by GPCR kinases (GRKs) that specifically phosphorylate the agonist-occupied form of the receptor. GRK-mediated receptor phosphorylation promotes the binding of arrestins, which function in GPCR desensitization, endocytosis and signaling. In this application, we propose to continue our studies aimed at further defining the mechanistic basis of GRK regulation of cell signaling. The first two aims will test the hypothesis that GRKs play an important role in transcriptional regulation via their ability to phosphorylate and regulate key cytoplasmic (aim 1) and nuclear (aim 2) substrates. The third aim will utilize C. elegans as a model organism to correlate GRK structure/function with biology. The specific objectives of the proposed studies are to: 1. Characterize the role of GRKs in NF-kB signaling. GRKs have now been shown to phosphorylate a variety of non-receptor substrates including the NF-kB protein p105. We propose to further characterize the mechanism and functional role of GRK5 phosphorylation of p105 and identify the signaling pathways that regulate this phosphorylation. These studies will provide important insight into the mechanisms that regulate NF-kB signaling and provide a potential link between GPCRs, GRKs and the NF-kB pathway. 2. Elucidate the role of GRKs in the nucleus. While GRKs have been implicated in the regulation of plasma membrane and cytoplasmic proteins, recent studies reveal that some GRKs can also localize in the nucleus. We will test the hypothesis that GRKs phosphorylate and regulate the function of specific nuclear substrates. We propose to identify the mechanism of GRK localization in the nucleus, identify the nuclear substrates of GRKs, and determine the functional role of GRK phosphorylation of selected substrates. 3. Characterize the functional role of GRKs in C. elegans. C. elegans is an excellent model to better understand the biological role of GRKs since the genome encodes ~1500 GPCRs but only 2 GRKs. In an effort to further define the role of GRKs and correlate structural features with in vivo function, we propose to analyze GRK-1, GRK-2, and GRK-1/GRK-2 deletion strains in a variety of biological assays including development, egg laying, growth, chemosensation, and longevity. Genetic crosses will be used to localize the GRKs in specific signaling pathways and these findings will be confirmed by biochemical analysis. These studies should enable us to define the biological role of the GRKs in C. elegans and address the role of specific protein interactions in GRK function. Overall, these studies should more clearly define the molecular and biological role of GRKs in regulating cell signaling and provide insight into the role of this important protein family in human disease. The Public Relevance: G protein-coupled receptor kinases play an important role in regulating cell signaling and have been implicated in a number of human diseases including heart failure and hypertension. Our proposed studies should more clearly define the molecular and biological role of GRKs in regulating cell signaling and provide insight into the role of this important protein family in human disease.